An active oxygen responsive hydrogen sulfide donor, an active oxygen responsive hydrogen sulfide controllable release type cationized chitosan sponge, a preparation method and application thereof

Abstract

This invention provides a reactive oxygen species (ROS)-responsive hydrogen sulfide donor, an ROS-responsive hydrogen sulfide-controlled release cationic chitosan sponge, its preparation method, and its applications, belonging to the field of medical materials. The ROS-responsive hydrogen sulfide-controlled release cationic chitosan sponge, prepared from quaternized chitosan and an ROS-responsive hydrogen sulfide donor, can controllably and on-demand release hydrogen sulfide under ROS stimulation. It can both reduce intracellular ROS levels and increase hydrogen sulfide levels. By inhibiting NF-κB activation, it regulates macrophage polarization towards the M2 phenotype, reduces the expression of inflammatory factors and eliminates excessive inflammation, enhances the migration and angiogenesis of human umbilical vein endothelial cells, and accelerates diabetic wound healing by promoting re-epithelialization, collagen deposition, and angiogenesis while simultaneously reducing inflammatory responses. The prepared ROS-responsive hydrogen sulfide-controlled release cationic chitosan sponge provides a promising solution for diabetic wound repair.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical materials technology, and in particular to a reactive oxygen species-responsive hydrogen sulfide donor, a reactive oxygen species-responsive hydrogen sulfide-controlled release cationic chitosan sponge, its preparation method, and its application. Background Technology

[0002] Diabetic foot is a difficult-to-treat chronic complication of diabetes, causing immense suffering and a heavy economic burden for patients. Excessive inflammation and impaired angiogenesis in wounds caused by excessive accumulation of reactive oxygen species (ROS), along with severe bacterial infection, are the main factors delaying wound healing. Macrophages are innate immune cells that play a crucial role in host defense, immune regulation, and wound healing. They can polarize into pro-inflammatory M1 macrophages and anti-inflammatory, pro-healing M2 macrophages. Excessive inflammation leads to a dysfunction in the polarization of macrophages from the M1 pro-inflammatory phenotype to the M2 anti-inflammatory, pro-healing phenotype, hindering the transition of wound healing from the inflammatory phase to the cell proliferation and tissue remodeling phase. Impaired angiogenesis obstructs blood circulation at the wound site, hindering the delivery of oxygen and nutrients and the removal of metabolic waste products, thus impeding the normal stromal cell function in wound repair. Bacterial infection leads to excessive infiltration of inflammatory cells, excessive production of ROS, and elevated levels of inflammatory cytokines, all of which hinder wound healing. Therefore, developing a bioactive dressing that integrates enhanced immune regulation, pro-angiogenesis, and anti-infection functions is an effective solution for promoting the repair of diabetic wounds.

[0003] Quaternized chitosan, a cationic chitosan derivative, has attracted widespread attention due to its inherent antibacterial activity, hemostatic potential, wound-healing ability, and good biocompatibility. Currently, various quaternized chitosan-based dressings have been developed for wound repair. Although these dressings can promote wound healing by inhibiting bacterial infection, their ability to eliminate oxidative stress and excessive inflammation, and to promote angiogenesis is limited, resulting in suboptimal wound repair outcomes.

[0004] Hydrogen sulfide is an endogenous gaseous signaling molecule with multiple biological activities. It can regulate macrophage polarization towards the M2 phenotype by inhibiting NF-κB activation, thereby eliminating excessive inflammation. Hydrogen sulfide can also alleviate oxidative stress in wounds by scavenging reactive oxygen species. Furthermore, it can promote reepithelialization, collagen deposition, and angiogenesis, thus accelerating wound healing. Currently, various small-molecule hydrogen sulfide donors (sodium hydrosulfide, sodium sulfide, and JK1) have been developed and integrated into materials for use in wound dressings. Despite their therapeutic potential, challenges remain regarding the controlled and on-demand release of hydrogen sulfide and its biocompatibility. Therefore, it is essential to develop a stimulus-responsive dressing that achieves controlled and on-demand release of hydrogen sulfide while ensuring good biocompatibility. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a reactive oxygen species (ROS) responsive hydrogen sulfide donor, a ROS-responsive hydrogen sulfide-controlled release cationic chitosan sponge, a preparation method therefor, and its applications. The ROS-responsive hydrogen sulfide donor provided by this invention can be covalently coupled to the surface of a QCC sponge, thereby obtaining a ROS-responsive hydrogen sulfide-controlled release cationic chitosan sponge for use in diabetic wound repair.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a reactive oxygen species-responsive hydrogen sulfide donor having the structure shown in Formula I:

[0008] Formula I.

[0009] This invention also provides a method for preparing the reactive oxygen species-responsive hydrogen sulfide donor described in the above technical solution, comprising the following steps:

[0010] The reaction of p-4-bromomethylphenylboronic acid pinacol ester with thiourea yields compound 1;

[0011] Compound 1 was subjected to a substitution reaction with 2,2′-dithiodipyridine to obtain compound 2;

[0012] The compound 2 was reacted with 3-mercaptopropionic acid to obtain the reactive oxygen species-responsive hydrogen sulfide donor.

[0013] Compound 1 has the structure shown in Formula 1, and compound 2 has the structure shown in Formula 2:

[0014] Formula 1 Formula 2.

[0015] The present invention also provides the application of the reactive oxygen species-responsive hydrogen sulfide donor described in the above technical solution in the preparation of wound dressings.

[0016] The present invention also provides a reactive oxygen species-responsive, hydrogen sulfide-controlled release cationic chitosan sponge having the structure shown in Formula II:

[0017] Formula II,

[0018] The Mw of the reactive oxygen species-responsive, hydrogen sulfide-controlled release cationic chitosan sponge is 90~110 kDa.

[0019] This invention also provides a method for preparing the reactive oxygen species-responsive hydrogen sulfide-controlled release cationic chitosan sponge described above, comprising the following steps:

[0020] The quaternized chitosan / chitosan solution was precooled to -20 °C and then freeze-dried to obtain quaternized chitosan / chitosan sponge, i.e. QCC sponge;

[0021] By an amidation reaction, a hydrogen sulfide donor is coupled to the surface of the QCC sponge to obtain the reactive oxygen species-responsive hydrogen sulfide-controlled release cationic chitosan sponge, namely, hydrogen sulfide / QCC sponge or H2S / QCC sponge, wherein the hydrogen sulfide donor is the reactive oxygen species-responsive hydrogen sulfide donor described in the above technical solution.

[0022] Preferably, the mass ratio of quaternized chitosan to chitosan in the quaternized chitosan / chitosan solution is 1:1.

[0023] Preferably, the ratio of the total mass of quaternized chitosan to chitosan and the volume of the solvent in the quaternized chitosan / chitosan solution is 3 g : 100 mL, and the solvent is an aqueous acetic acid solution with a concentration of 2 vol.

[0024] Preferably, the molar ratio of the carboxyl group in the reactive oxygen species-responsive hydrogen sulfide donor to the amino group in the QCC sponge is not less than 1.

[0025] The present invention also provides the application of the reactive oxygen species-responsive hydrogen sulfide-controlled release cationic chitosan sponge described above in the preparation of wound dressings.

[0026] Preferably, the wound dressing is used for the repair of diabetic wounds.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] This invention provides a reactive oxygen species (ROS)-responsive cationic chitosan sponge with controlled release of hydrogen sulfide, composed of quaternized chitosan, chitosan, and an ROS-responsive hydrogen sulfide donor. The ROS-responsive hydrogen sulfide donor is covalently coupled to the surface of the QCC sponge, which improves the bioavailability of hydrogen sulfide while ensuring good biocompatibility. Upon exposure to ROS, the borate ester protecting groups in the donor detach, scavenging the ROS and initiating spontaneous breakage of self-degrading bonds, thereby releasing hydrogen sulfide.

[0029] The released hydrogen sulfide reduces oxidative stress by consuming reactive oxygen species; it promotes M2 polarization of Raw264.7 macrophages by inhibiting NF-κB activation, thereby reducing the expression of inflammatory factors and eliminating excessive inflammation; and it enhances the migration and angiogenesis of human umbilical vein endothelial cells by upregulating the expression of VEGF and eNOS. The quaternized chitosan in the sponge matrix exhibits strong antibacterial / anti-infective activity through electrostatic interactions with *Escherichia coli* and methicillin-resistant *Staphylococcus aureus*, effectively reducing wound inflammation. Furthermore, this reactive oxygen species-responsive, hydrogen sulfide-controlled release cationic chitosan sponge can accelerate diabetic wound healing by promoting re-epithelialization, collagen deposition, and angiogenesis while reducing inflammatory responses, demonstrating superior efficacy compared to commercially available 3M products. TM Dressings. The reactive oxygen species-responsive cationic chitosan sponge with controlled hydrogen sulfide release prepared in this invention not only solves the problem of existing hydrogen sulfide dressings being unable to achieve controlled and on-demand release of hydrogen sulfide, but also provides a promising solution for diabetic wound repair. Attached Figure Description

[0030] Figure 1 The images show the preparation and characterization of hydrogen sulfide / QCC sponges. A represents the preparation process of the hydrogen sulfide / QCC sponge; B is the attenuated total reflectance Fourier transform infrared spectrum; C is the proton NMR spectrum; D is the X-ray photoelectric spectrum; E is the energy dispersive spectroscopy (EDS) analysis; F is the distribution of sulfur (S) and boron (B); G is the water adsorption rate; H is the scanning electron microscope (SEM) image; I is the compressive stress-strain curve; J is the hydrogen sulfide release-time curve; K is the hydrogen peroxide consumption-time curve; L is the relative expression level of reactive oxygen species (ROS) / hydrogen sulfide (H2S) in lipopolysaccharide-stimulated Raw 264.7 macrophages after sponge treatment; and M is the fluorescence image of reactive oxygen species (ROS) / hydrogen sulfide (H2S) in lipopolysaccharide-stimulated Raw 264.7 macrophages after sponge treatment.

[0031] Figure 2 The images show the biocompatibility of hydrogen sulfide / QCC sponges. A represents the hemolysis rate in the water, saline, QCC sponge, and hydrogen sulfide / QCC sponge groups, with the inset showing a hemolysis test photograph. B is the optical density map of human umbilical vein endothelial cells (HUVECs) co-cultured with hydrogen sulfide / QCC sponges. C is the optical density map of Raw 264.7 macrophages co-cultured with hydrogen sulfide / QCC sponges. D is the live / dead staining image of HUVECs and Raw 264.7 macrophages co-cultured with hydrogen sulfide / QCC sponges. E-H represent the results of complete blood count and serum biochemical tests.

[0032] Figure 3The results of the hemostatic potential test are as follows: A is a digital photograph of the untreated wound and the wound treated with gelatin sponge, QCC sponge and hydrogen sulfide / QCC sponge in the rat femoral artery injury model; B is the total blood loss in different groups in the rat femoral artery injury model; C is the blood solidification index; and D is a scanning electron microscope (SEM) image of blood cell adhesion.

[0033] Figure 4 The results are as follows: A shows the cytoskeleton staining image of lipopolysaccharide-stimulated Raw 264.7 macrophages after treatment with QCC and hydrogen sulfide / QCC sponge; B shows the immunofluorescence image of CD86 and CD206 in lipopolysaccharide-stimulated Raw 264.7 macrophages after treatment with QCC and hydrogen sulfide / QCC sponge; C shows the flow cytometry image of CD86 and CD206 expression; D and E show the expression levels of CD86 and CD206; F, G, and H show the expression levels of IL-1β, CD163, and TNF-α in lipopolysaccharide-stimulated Raw 264.7 macrophages after treatment with QCC and hydrogen sulfide / QCC sponge; I shows the Western blot image of CD206, CD86, and NF-κB expression in different groups; and J shows the relative expression levels of CD206, CD86, and NF-κB in different groups.

[0034] Figure 5 The results are as follows: A shows the migration images of human umbilical vein endothelial cells (HUVECs) at 0, 12, and 24 h; B and C show the scratch healing rate at 12 and 24 h; D shows the quantitative data (cell count) of the Transwell assay; E shows the Transwell images of human umbilical vein endothelial cells (HUVECs) stimulating with hydrogen peroxide (H2O2) after treatment with QCC and hydrogen sulfide / QCC sponge extract, migrating to the lower chamber.

[0035] Figure 6 The results of the angiogenesis test of the sponge are shown in Figure A, where A is an image of tubular structures formed by human umbilical vein endothelial cells (HUVECs) stimulated by hydrogen peroxide (H2O2) after treatment with QCC and hydrogen sulfide / QCC sponge extract; B is the number of tubular structures in different groups; C and D are the relative expression levels of VEGF and eNOS; and E is the fluorescence image of VEGF and eNOS expression.

[0036] Figure 7The results are as follows: A shows the antibacterial activity test results, where A represents the antibacterial rate of hydrogen sulfide / QCC sponge against Escherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA); B shows digital images of bacterial colony units in the control group and the hydrogen sulfide / QCC sponge group; C shows fluorescent staining images of live / dead bacteria in the control group and the hydrogen sulfide / QCC sponge group; D shows digital images of bacterial colony units in a rat skin full-thickness defect model infected with bacteria; E shows hematoxylin-eosin (H&E) staining images of bacterial-infected wounds in the untreated and hydrogen sulfide / QCC sponge treated groups; and F shows immunofluorescence staining images of CD86, IL-6, and iNOS in the untreated group and the hydrogen sulfide / QCC sponge group.

[0037] Figure 8 The results of skin wound repair for diabetic patients, where A represents 3M. TM Digital images of wound treatment with dressings, QCC sponges, and hydrogen sulfide / QCC sponges; B is a schematic diagram of wound area at different time points; C is the wound healing rate of different groups; D is the hematoxylin-eosin (H&E) staining images of wounds in different treatment groups 7 days postoperatively; E is the Masson trichrome staining images of wounds in different treatment groups 7 days postoperatively; F, G, and H are the immunofluorescence staining images of CD86, CD206, and CD31 in different treatment groups 7 days postoperatively, respectively.

[0038] Figure 9 This diagram illustrates the working principle of a reactive oxygen species (ROS) responsive cationic chitosan sponge with controlled hydrogen sulfide release. A represents the mechanism by which the ROS-responsive cationic chitosan sponge responds to and releases hydrogen sulfide in a controlled manner; B represents the potential application and mechanism of this ROS-responsive cationic chitosan sponge in diabetic wound repair. Detailed Implementation

[0039] This invention provides a reactive oxygen species-responsive hydrogen sulfide donor having the structure shown in Formula I:

[0040] Formula I.

[0041] This invention also provides a method for preparing the reactive oxygen species-responsive hydrogen sulfide donor described in the above technical solution, comprising the following steps:

[0042] The reaction of p-4-bromomethylphenylboronic acid pinacol ester with thiourea yields compound 1;

[0043] Compound 1 was subjected to a substitution reaction with 2,2′-dithiodipyridine to obtain compound 2;

[0044] The compound 2 was subjected to a mercapto-exchange reaction with 3-mercaptopropionic acid to obtain the reactive oxygen species-responsive hydrogen sulfide donor.

[0045] Compound 1 has the structure shown in Formula 1, and compound 2 has the structure shown in Formula 2:

[0046] Formula 1 Formula 2.

[0047] In this invention, the preparation principle of the reactive oxygen species-responsive hydrogen sulfide donor is shown in the following formula:

[0048] .

[0049] In this invention, 4-bromomethylphenylboronic acid pinacol ester is reacted with thiourea to obtain compound 1.

[0050] In this invention, the preferred mass ratio of the 4-bromomethylphenylboronic acid pinacol ester to thiourea is 3:1.

[0051] In this invention, the CAS number of the 4-bromomethylphenylboronic acid pinacol ester is 138500-85-3.

[0052] In this invention, the reaction is preferably carried out under the protection of a solvent and nitrogen, and the solvent is preferably anhydrous ethanol.

[0053] In this invention, the reaction temperature is preferably room temperature, without the need for additional heating or cooling, and the reaction time is preferably 4 to 6 hours.

[0054] In this invention, the 4-bromomethylphenylboronic acid pinacol ester is dissolved in anhydrous ethanol, and under nitrogen protection, the thiourea is added to react. The reactants are then concentrated by rotary evaporation, and the resulting thiourea intermediate is dissolved in an aqueous sodium hydroxide solution and refluxed for 1 h. The resulting mixture is cooled in an ice bath, and hydrochloric acid is added to form a white precipitate. The aqueous phase is then extracted, the organic phase is separated and dried, and the crude product is purified by silica gel column chromatography to obtain compound 1.

[0055] After obtaining compound 1, the present invention performs a substitution reaction between compound 1 and 2,2′-dithiopyridine to obtain compound 2.

[0056] In this invention, the preferred mass ratio of compound 1 to 2,2′-dithiopyridine is 1:1.8.

[0057] In this invention, the substitution reaction is preferably carried out in a solvent, which preferably includes anhydrous methanol.

[0058] In this invention, the temperature of the substitution reaction is preferably room temperature, without the need for additional heating or cooling, and the time is preferably 10-12 h.

[0059] In this invention, compound 1 is preferably dissolved in anhydrous methanol and reacted for 12 h. After the product is concentrated, the crude product is purified by silica gel column chromatography to obtain compound 2.

[0060] After obtaining compound 2, the present invention performs a thiol exchange reaction between compound 2 and 3-mercaptopropionic acid to obtain the reactive oxygen species responsive hydrogen sulfide donor.

[0061] In this invention, the preferred mass ratio of compound 2 to 3-mercaptopropionic acid is 1:0.3.

[0062] In this invention, the thiol exchange reaction is preferably carried out in a solvent, which preferably includes anhydrous methanol.

[0063] In this invention, the temperature of the thiol exchange reaction is preferably room temperature, without the need for additional heating or cooling, and the time is preferably 4 to 6 hours.

[0064] In this invention, compound 2 and 3-mercaptopropionic acid are preferably dissolved in anhydrous methanol and reacted for 4-6 h. After the product is concentrated, the crude product is purified by silica gel column chromatography to obtain the reactive oxygen species-responsive hydrogen sulfide donor.

[0065] The present invention also provides the application of the reactive oxygen species-responsive hydrogen sulfide donor described in the above technical solution in the preparation of wound dressings.

[0066] The present invention also provides a reactive oxygen species-responsive, hydrogen sulfide-controlled release cationic chitosan sponge having the structure shown in Formula II:

[0067] Formula II,

[0068] The Mw of the reactive oxygen species-responsive hydrogen sulfide-controlled release cationic chitosan sponge is 90~110 kDa, specifically 100 kDa.

[0069] This invention also provides a method for preparing the reactive oxygen species-responsive hydrogen sulfide-controlled release cationic chitosan sponge described above, comprising the following steps:

[0070] The quaternized chitosan / chitosan solution was precooled to -20 °C and then freeze-dried to obtain quaternized chitosan / chitosan sponge, i.e. QCC sponge;

[0071] By an amidation reaction, a hydrogen sulfide donor is coupled to the surface of the QCC sponge to obtain the reactive oxygen species-responsive hydrogen sulfide-controlled release cationic chitosan sponge, namely, hydrogen sulfide / QCC sponge or H2S / QCC sponge, wherein the hydrogen sulfide donor is the reactive oxygen species-responsive hydrogen sulfide donor described in the above technical solution.

[0072] In this invention, a quaternized chitosan / chitosan solution is precooled to -20 °C and then freeze-dried to obtain a QCC sponge.

[0073] In this invention, the quaternized chitosan is preferably prepared by a method comprising the following steps:

[0074] Chitosan was dispersed in an aqueous acetic acid solution to obtain a chitosan suspension;

[0075] 2,3-epoxypropyltrimethylammonium chloride was dissolved in an aqueous acetic acid solution to obtain a 2,3-epoxypropyltrimethylammonium chloride solution;

[0076] The 2,3-epoxypropyltrimethylammonium chloride solution was added to the chitosan suspension and reacted at 55 °C for 24 h. The reaction solution was centrifuged to obtain the supernatant, which was then placed in a dialysis bag and dialyzed in water for 3 days. After freeze-drying, the quaternized chitosan was obtained.

[0077] In this invention, the preparation principle of the quaternized chitosan is shown in the following formula:

[0078] .

[0079] In this invention, the preferred mass ratio of chitosan to 2,3-epoxypropyltrimethylammonium chloride is 4:6.4.

[0080] In this invention, the concentration of the aqueous acetic acid solution is preferably 0.5 vol%.

[0081] In a specific embodiment of the present invention, preferably, 4 g of the chitosan is dispersed in 144 mL of acetic acid aqueous solution, 6.4 g of the 2,3-epoxypropyltrimethylammonium chloride is dissolved in 40 mL of acetic acid aqueous solution, the resulting 2,3-epoxypropyltrimethylammonium chloride solution is added to the chitosan suspension, and after reacting at 55 °C for 24 h, the mixture is centrifuged, the supernatant is taken, dialyzed with deionized water and freeze-dried to obtain the quaternized chitosan.

[0082] In this invention, the preferred mass ratio of quaternized chitosan to chitosan in the quaternized chitosan / chitosan solution is 1:1.

[0083] In this invention, the ratio of the total mass of quaternized chitosan and chitosan to the solvent volume in the quaternized chitosan / chitosan solution is preferably 3 g: 100 mL, the solvent is preferably an aqueous acetic acid solution, and the concentration of acetic acid in the aqueous acetic acid solution is preferably 2 vol.

[0084] In this invention, the quaternized chitosan / chitosan solution is preferably centrifuged and then cooled to -20 °C for freeze drying.

[0085] In this invention, the washing is preferably performed by sequentially washing with sodium hydroxide / ethanol solution and water. This invention does not impose any special limitations on the specific parameters of the washing, and any method known to those skilled in the art can be used.

[0086] After the washing process is completed, the present invention preferably performs freeze drying to obtain the QCC sponge.

[0087] After obtaining the QCC sponge, the present invention couples a hydrogen sulfide donor to the surface of the QCC sponge through an amidation reaction. The QCC sponge and the hydrogen sulfide donor are then mixed and subjected to an amidation reaction to obtain the reactive oxygen species-responsive hydrogen sulfide-controlled release cationic chitosan sponge, namely, hydrogen sulfide / QCC sponge or H2S / QCC sponge, wherein the hydrogen sulfide donor is the reactive oxygen species-responsive hydrogen sulfide donor described in the above technical solution.

[0088] In this invention, the molar ratio of the carboxyl group in the reactive oxygen species-responsive hydrogen sulfide donor to the amino group in the QCC sponge is not less than 1. In a specific embodiment of this invention, the mass ratio of the QCC sponge to the reactive oxygen species-responsive hydrogen sulfide donor is preferably 1:0.4.

[0089] In this invention, the amidation reaction is preferably carried out under the conditions of N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), the mass ratio of QCC sponge to EDC is preferably 1:1, and the mass ratio of QCC sponge to NHS is preferably 1:0.6.

[0090] In this invention, the temperature of the amidation reaction is preferably room temperature, and the time is preferably 48 h.

[0091] After the amidation reaction is completed, the resulting amidation product is preferably thoroughly washed, then cooled to -20 °C and freeze-dried to obtain a QCC sponge modified with a reactive oxygen species-responsive hydrogen sulfide donor, namely the reactive oxygen species-responsive hydrogen sulfide-controlled release cationic chitosan sponge.

[0092] The present invention also provides the application of the reactive oxygen species-responsive hydrogen sulfide-controlled release cationic chitosan sponge described in the above technical solution in the preparation of wound dressings.

[0093] In this invention, the wound dressing is preferably used for the repair of diabetic wounds.

[0094] Figure 9This diagram illustrates the working principle of a reactive oxygen species (ROS) responsive cationic chitosan sponge with controlled hydrogen sulfide release. A represents the mechanism by which the ROS-responsive cationic chitosan sponge responds to and releases hydrogen sulfide in a controlled manner. B represents the potential application and mechanism of this ROS-responsive cationic chitosan sponge in the repair of diabetic skin wounds.

[0095] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0096] Material

[0097] Chitosan (molecular weight 100 kDa, degree of deacetylation 85.3%) was purchased from Jinan Haidebei Marine Biotechnology Co., Ltd. 2,3-epoxypropyltrimethylammonium chloride, N-hydroxysuccinimide (NHS), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The reactive oxygen species (ROS) detection kit was purchased from Nanjing Jiancheng Biotechnology Institute. The hydrogen peroxide (H2O2) detection kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd. The SYTO9 / PI live / dead bacteria double staining kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd. The Calcein-AM / PI live / dead cytotoxicity assay kit, cell counting kit-8, hematoxylin-eosin (H&E) staining kit, Masson's trichrome staining kit, lipopolysaccharide, FITC-labeled phalloidin, WSP-5 probe, and 4,6-diamidinyl-2-phenylindole dihydrochloride (DAPI) were purchased from Beijing Solarbio Science & Technology Co., Ltd. The TNF-α, CD163, and IL-1β ELISA kits for mice were purchased from Shanghai Qifa Experimental Reagent Co., Ltd. Raw 264.7 macrophages were purchased from Wuhan Pronosei Biotechnology Co., Ltd. Human umbilical vein endothelial cells (HUVECs) were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. Sprague-Dawley rats (male, 300-320 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0098] Animal experiments were conducted in strict accordance with the guidelines of the U.S. National Research Council and approved by the Institutional Animal Care and Use Committee of Yishengyuan Gene Technology (Tianjin) Co., Ltd., with experiment number YSY-DWLL-2024759.

[0099] Characterization

[0100] By attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and proton nuclear magnetic resonance (NMR)1 The chemical structure of the sponge was characterized by ¹H NMR. The microstructure of the sponge was observed by scanning electron microscopy (SEM). The elemental distribution of carbon (C), sulfur (S), and boron (B) in the sponge was detected by energy dispersive spectroscopy (EDS). Fine spectra of S and B in the sponge were obtained by X-ray photoelectron spectroscopy (XPS). The compressive strength of a cylindrical sponge (10 mm in diameter and 10 mm in height) was tested using a food texture analyzer at 60% strain and a rate of 50 mm / min. The dry sponge was immersed in 30 mL of deionized water. After reaching adsorption equilibrium, the surface moisture of the sponge was gently absorbed and its weight was weighed. The water absorption rate of the sponge was calculated according to formula (1):

[0101] Formula (1)

[0102] In the formula, W1 and W2 represent the weight of the sponge before and after absorbing water, respectively.

[0103] Hydrogen sulfide release test

[0104] Hydrogen sulfide / QCC sponge (10 mg) was immersed in 2 mL of phosphate-buffered saline (PBS, pH = 7.4) with or without hydrogen peroxide (100 µM). At regular time intervals, 50 µL of the supernatant was transferred to a 96-well plate, followed by the addition of Zn(COO)₂ solution (10 μL, 1 wt%), ferric chloride solution dissolved in 1.2 M hydrochloric acid (20 μL, 30 mM), and N,N-dimethyl-p-phenylenediamine solution dissolved in 7.2 M hydrochloric acid (20 μL, 20 mM). After 20 min, the optical density at 670 nm was measured using a microplate reader (BIORAD, USA).

[0105] Hydrogen peroxide scavenging experiment

[0106] Immerse a sponge (10 mg) in 2 mL of PBS solution containing hydrogen peroxide (100 µM). At regular time intervals, transfer 50 µL of the supernatant to a 96-well plate and mix with 100 μL of the detection reagent from the hydrogen peroxide detection kit. After 30 min, measure the optical density at 560 nm using a microplate reader.

[0107] Intracellular reactive oxygen species scavenging experiment

[0108] Raw 264.7 macrophages (4 × 10⁻⁶) 4Cells were seeded in 24-well plates and incubated at 37 °C for 24 h. Cells were stimulated with 100 ng / mL lipopolysaccharide solution. After 24 h, 5 mg sponges were placed in the upper chamber of a Transwell plate for co-incubation with the cells. After 24 h, 500 μL of probe working solution (10 µM) was added to each well. After 40 min, the solution was removed, and the plates were washed with PBS. Hoechst 33342 staining solution was then added and incubated for 5 min. The staining solution was removed, the plates were washed with PBS, and imaging was performed using an inverted fluorescence microscope (CKV53-LP).

[0109] Intracellular hydrogen sulfide release experiment

[0110] Raw 264.7 macrophages (5 × 10⁻⁶) 5 Cells were seeded in 12-well plates and incubated at 37 °C for 12 h. Cells were stimulated with 100 ng / mL lipopolysaccharide solution. After 2 h, 5 mg sponges were placed in the upper chamber of a Transwell plate for co-incubation with the cells. After 6 h, WSP-5 probe solution (50 μM) and Hoechst 33342 staining solution (10 μg / mL) were added to the wells for co-incubation with the cells. After 30 min, the cells were washed with PBS and imaged using an inverted fluorescence microscope.

[0111] Cytoskeleton staining

[0112] Raw 264.7 macrophages (2.5 × 10⁻⁶) 5 M1 macrophages were seeded in 24-well plates and incubated at 37 °C for 48 h. M2 macrophages were polarized by adding 100 ng / mL lipopolysaccharide solution, while M2 macrophages were polarized by adding 20 ng / mL IL-4 / IL-13 solution. After 24 h, 5 mg of sponge was placed in the upper chamber of a Transwell plate for co-incubation with the cells. After 24 h, the cells were fixed and permeabilized with paraformaldehyde and Triton X-100 solution, respectively. The cytoskeleton and nucleus were then stained with FITC-labeled phalloidin and DAPI, respectively. The cells were imaged using an inverted fluorescence microscope.

[0113] Immunofluorescence staining

[0114] Raw 264.7 macrophages were loaded at 4 × 10⁶ cells per well. 4Cells were seeded at a density of 100 g / mL in 24-well plates and cultured at 37 °C for 24 h. Cells were stimulated with 100 ng / mL lipopolysaccharide solution. After 24 h, 5 mg sponges were placed in the upper chamber of a Transwell plate for co-incubation with the cells. After 24 h, the culture medium was removed, and cells were fixed, permeabilized, and blocked using paraformaldehyde, Triton X-100, and bovine serum albumin solutions, respectively. Subsequently, diluted CD206 monoclonal antibody (1:200, HUABIO) or CD86 polyclonal antibody (1:500, Proteintech Group) was added to the wells. After overnight co-incubation at 4 °C, diluted FITC-labeled goat anti-rabbit IgG (H+L) antibody (1:500, Beyotime) and DAPI were added to the wells for 1 h and 5 min, respectively. Cells were imaged using an inverted fluorescence microscope.

[0115] Flow cytometry analysis

[0116] Raw 264.7 macrophages (4 × 10⁻⁶) 4 Cells were seeded in 24-well plates and cultured at 37 °C for 24 h. Cells were stimulated with 100 ng / mL lipopolysaccharide solution. After 24 h, 5 mg sponges were placed in the upper chamber of a Transwell plate for co-incubation with the cells. After 24 h, cells were collected, blocked with anti-mouse CD16 / 32 antibody (Invitrogen), centrifuged, and resuspended in PBS. FITC-labeled anti-mouse CD86 antibody (Elabscience) was added and incubated with the cells for 30 min. Subsequently, the cells were fixed and permeabilized with paraformaldehyde and Triton X-100 solutions, respectively. The cells were resuspended in PBS again and incubated with PE-labeled anti-mouse CD206 antibody (BioLegend) for 30 min. After washing, the cells were resuspended in PBS and analyzed by flow cytometry (NovoCyte 2000).

[0117] Cytokine detection

[0118] Raw 264.7 macrophages (4 × 10⁻⁶) 4 Cells were seeded in 24-well plates and incubated at 37 °C for 24 h. Cells were stimulated with 100 ng / mL lipopolysaccharide solution. After 24 h, 5 mg of sponge was placed in the upper chamber of a Transwell plate for co-incubation with the cells. After 24 h, the levels of cytokines (TNF-α, IL-1β, and CD163) in the supernatant were measured using an ELISA kit.

[0119] Western blot analysis

[0120] The expression levels of CD86, CD206, and NF-κB in Raw 264.7 macrophages stimulated with lipopolysaccharide solution were detected using standard Western blotting. The following antibodies were used in the experiment: CD206 antibody (1:1000, HUABIO), CD86 polyclonal antibody (1:1000, HUABIO), NF-κB antibody (1:5000, HUABIO), mouse anti-human microtubule (β) antibody (1:3000, ZSGB-Bio), goat anti-rabbit IgG antibody (1:5000, ZSGB-Bio), and goat anti-mouse IgG antibody (1:5000, ZSGB-Bio).

[0121] Cell scratch assay

[0122] Human umbilical vein endothelial cells (HUVECs) (5 × 10⁻⁶) 5 Cells were seeded in 6-well plates and cultured at 37 °C for 48 h. After complete cell spread, the cells were stimulated with 100 μM hydrogen peroxide solution. Two h later, scratches were made using a 200 μL pipette tip, followed by washing with PBS and co-incubation with sponge extract. Cell migration images were acquired using a fluorescence microscope at 0, 12, and 24 h, and the scratch healing rate was calculated using formula (2):

[0123] Formula (2)

[0124] In the formula, S1 and S2 represent the scratch area at 0 h and 12 or 24 h, respectively.

[0125] Cell invasion assay

[0126] 5 × 10 3 Personal umbilical vein endothelial cells (HUVECs) were seeded on the surface of the matrix gel in the upper chamber of a Transwell. 100 μM H₂O₂ and sponge extraction medium were added to the lower chamber of the Transwell. After 24 h, the culture medium was removed, and the upper chamber was washed with PBS. Crystal violet staining solution was then added to the upper chamber to stain the cells. After 5 min, unmigrated cells were removed from the upper chamber. Migrating cells were observed and imaged using an inverted microscope.

[0127] Tube Formation Test

[0128] Human umbilical vein endothelial cells (HUVECs) were added at a rate of 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 100 μM on the surface of a 96-well plate using a matrix gel. The cells were stimulated with 100 μM hydrogen peroxide solution, and after 2 h, sponge extract was added to the wells for co-incubation for 6 h. The cells were then imaged using a microscope.

[0129] VEGF and eNOS expression levels

[0130] 2 × 10 4 Personal umbilical vein endothelial cells (HUVECs) were seeded onto cell crawling slides within 24-well plates and incubated at 37 °C for 24 h. Subsequently, cells were stimulated with 100 μM H₂O₂ for 2 h and co-cultured with 5 mg sponges placed in the upper chamber of a Transwell. After 24 h, the culture medium was removed, and cells were fixed, permeabilized, and blocked sequentially with paraformaldehyde, Triton X-100, and bovine serum albumin. Blocked cells were then incubated overnight at 4 °C in the dark with diluted rabbit recombinant anti-vascular endothelial growth factor (VEGF) monoclonal antibody (1:100, HUABIO) or anti-endothelial nitric oxide synthase (eNOS) antibody (1:100, HUABIO). Cells were thoroughly washed with PBS and then co-incubated with FITC-labeled goat anti-rabbit IgG (H+L) secondary antibody (1:500, Beyotime). After 30 min, the cells were washed again with PBS, the nuclei were stained with DAPI, and the images were taken using an inverted fluorescence microscope.

[0131] Rat femoral artery hemostasis

[0132] Rats were anesthetized with chloral hydrate and fixed on the operating table. The femoral artery was exposed through an incision. Bleeding was induced by puncturing the femoral artery with a 26G needle. After bleeding, a sponge (gelatin sponge, QCC sponge, or hydrogen sulfide / QCC sponge) was immediately applied to the bleeding site. Gauze was used to absorb the oozing blood from the wound. After hemostasis, the bleeding site was photographed, and the total blood loss was calculated by weighing.

[0133] Blood solidification index test

[0134] 120 μL of sodium citrate-treated whole blood (whole blood to 0.1 M calcium chloride solution volume ratio 10:2) was co-incubated with a sponge at 37 ℃ for 5 min. Then, 2 mL of deionized water was added to lyse red blood cells and release hemoglobin. After 5 min, 100 μL of the supernatant was taken and its optical density at 540 nm was measured. Sodium citrate-treated whole blood that was not co-incubated with the material was used as a control. The blood solidification index was calculated according to formula (3):

[0135] Formula (3)

[0136] In the formula, OD s and OD c These represent the optical density values ​​of the sponge group and the control group, respectively.

[0137] Blood cell adhesion assay

[0138] 300 μL of sodium citrate-anticoagulated whole blood was added to the surface of a sponge and incubated at 37 °C for 1 h. The sponge was washed with physiological saline to remove unadhered blood cells, and then immersed in a 2.5 vol% glutaraldehyde solution. After 2 h, the sponge was dehydrated using a gradient ethanol solution. After freeze-drying, the sponge was sputter-coated with gold, and then observed and imaged using a scanning electron microscope.

[0139] Antibacterial activity evaluation

[0140] A suspension of methicillin-resistant Staphylococcus aureus (MRSA) or Escherichia coli (E. coli) (10) 7 CFU / mL) was co-incubated with hydrogen sulfide / QCC sponge at 37 °C for 2 h. The viable bacteria were resuspended in PBS. The resuspended solution was inoculated onto an agar plate. After 24 h, colony units were counted. The antibacterial rate was calculated using formula (4):

[0141] Formula (4)

[0142] In the formula, A c and A s These represent the number of colony units in the control group (agar plate alone) and the sponge group, respectively.

[0143] A suspension of methicillin-resistant Staphylococcus aureus (MRSA) or Escherichia coli (E. coli) (10) 7 The bacteria (CFU / mL) were co-incubated with hydrogen sulfide / QCC sponges at 37 °C for 2 h. The live bacteria were resuspended in PBS. The sponge was immersed in 2 mL of PBS and centrifuged to collect the bacterial pellet. The bacteria were resuspended in 200 μL of SYTO 9 / PI staining solution. After 15 min, 5 μL of the resuspended solution was added to a glass slide and imaged using a fluorescence microscope.

[0144] Rats were anesthetized with chloral hydrate and fixed to the operating table. Hair was removed from the back area, and the area was disinfected with povidone-iodine. A circular, full-thickness skin defect was created on each side of the back using a tissue biopsy device. A suspension of methicillin-resistant Staphylococcus aureus (MRSA) or Escherichia coli (E. coli) (10...) was then placed in the surgical area. 7 CFU / mL was applied to the wound surface. The wound was then covered with a hydrogen sulfide / QCC sponge. Two days post-surgery, wound tissue was collected for bacterial smearing and histological analysis. Hematoxylin-eosin (H&E) staining and immunofluorescence staining were used to evaluate inflammatory cell infiltration and the expression of inflammatory cytokines (CD86, IL-6, and iNOS).

[0145] Biocompatibility assessment

[0146] 1 × 104 264.7 macrophages per raw sample or 1 × 10⁻⁶ macrophages 6 Personal umbilical vein endothelial cells (HUVECs) were seeded in 48-well plates and cultured at 37 °C for 12 h, after which the culture medium was replaced with sponge extract. After co-incubation for 1 and 3 days, reagents were added to the wells. The optical density of the supernatant at 450 nm was measured 2 h later. Additionally, 1 × 10⁻⁶ HUVECs were... 4 264.7 macrophages per raw sample or 1 × 10⁻⁶ macrophages 6 Personal umbilical vein endothelial cells (HUVECs) were seeded onto cell spreaders embedded in 48-well plates. After overnight culture at 37 °C, the culture medium was replaced with sponge extract. After co-culturing for 1 and 3 days, the supernatant was removed, and Calcein-AM / PI working solution was added. After 30 min, the stained cells were imaged using a fluorescence microscope.

[0147] Red blood cells were obtained by centrifuging sodium citrate-anticoagulated whole blood at 3500 rpm for 10 min, and then resuspended in physiological saline to prepare a red blood cell suspension. 5 mg of sponge was immersed in 1 mL of the suspension. After co-incubation at 37 ℃ for 1 h, centrifugation was performed, and the supernatant was collected. The optical density at 540 nm was measured using an ELISA reader. The hemolysis rate was calculated according to formula (5):

[0148] Formula (5)

[0149] In the formula, L s L w and L n These represent the optical density values ​​for the sponge, water, and saline groups, respectively.

[0150] Rats were anesthetized with chloral hydrate. After removing hair from the back area and disinfecting with povidone-iodine, an incision was made on the rat's back. A circular sponge was implanted subcutaneously. Fourteen days post-surgery, the rats were anesthetized, and blood was collected for complete blood count and serum biochemical analysis.

[0151] Diabetic skin wound repair

[0152] A diabetic rat model was established by intraperitoneal injection of streptozotocin. Rats were anesthetized with chloral hydrate, and subsequently, their dorsal hair was shaved and the area disinfected with povidone-iodine. A 1 cm diameter full-thickness biopsy lesion was created on each side of the rat's back using a tissue biopsy instrument and covered with a sponge. A commercially available 3M... TM The dressing served as a control group. Macroscopic photographs of the wound were taken on postoperative days 5, 7, and 14, and the wound healing rate was calculated according to formula (6):

[0153] Formula (6)

[0154] In the formula, Sc and S s These represent sponge or 3M respectively. TM Wound area before and after dressing treatment.

[0155] Histological analysis

[0156] On postoperative day 7, wound tissue was collected and subjected to histological analysis. The specific procedures were as follows: First, the wound tissue was fixed in paraformaldehyde solution, then dehydrated sequentially with graded ethanol and xylene, embedded in paraffin, and cut into 5 μm thick sections. Hematoxylin and eosin (H&E) staining was used to evaluate the integrity of the tissue structure. Masson's trichrome staining was used to evaluate collagen deposition. Furthermore, CD31, CD86, and CD206 immunofluorescence staining were used to evaluate wound vascularization and inflammatory response, respectively.

[0157] Statistical analysis

[0158] All results are expressed as mean ± standard deviation. Statistical analyses were performed using GraphPad Prism 8 software via One-Way Analysis of Variance (ANOVA) with Tukey's multiple comparison test or unpaired t-test. P <0.05 indicates a statistically significant difference. P >0.05 indicates no statistically significant difference (ns).

[0159] Example 1

[0160] Synthesis and characterization of reactive oxygen species-responsive hydrogen sulfide donors

[0161] Under a nitrogen atmosphere, 3 g of 4-bromomethylphenylboronic acid pinacol ester was dissolved in 30 mL of anhydrous ethanol, followed by the addition of 1 g of thiourea. After reacting for 5 h, the reactants were concentrated by rotary evaporation to obtain a thiourea intermediate. The thiourea intermediate was dissolved in 0.1 g / mL sodium hydroxide solution and refluxed for 1 h. The reactants were cooled in an ice bath, and hydrochloric acid was added to form a white precipitate. The aqueous phase was extracted, the organic phase was separated, and dried to obtain a crude product. The crude product was purified by silica gel column chromatography to obtain compound 1. Compound 1 (1 g) and 2,2′-dithiodipyridine (1.8 g) were dissolved in 20 mL of anhydrous methanol and reacted for 12 h. The reactants were concentrated to obtain a crude product, which was purified by silica gel column chromatography to obtain compound 2. Compound 2 (1 g) and 0.3 g of 3-mercaptopropionic acid were dissolved in 10 mL of anhydrous methanol and reacted for 4 h. The reactants were concentrated to obtain a crude product, which was then purified by silica gel column chromatography to obtain a reactive oxygen species-responsive hydrogen sulfide donor. The donor was further purified by proton NMR spectroscopy. 1 The chemical structure of the donor was characterized using a 1H NMR instrument (Bruker AVII-400 MHz) with CDCl3 as the solvent.

[0162] Synthesis and characterization of quaternized chitosan

[0163] 4 g of chitosan was dispersed in 144 mL of 0.5 vol% acetic acid aqueous solution. 6.4 g of 2,3-epoxypropyltrimethylammonium chloride was dissolved in 40 mL of 0.5 vol% acetic acid aqueous solution. The 2,3-epoxypropyltrimethylammonium chloride solution was added to the chitosan suspension. After reacting at 55 °C for 24 h, the reaction mixture was centrifuged to obtain the supernatant, which was then placed in a dialysis bag and dialyzed against deionized water for 3 days. After freeze-drying, quaternized chitosan was obtained. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) using a Thermo Fisher IS5 and proton nuclear magnetic resonance (NMR) were employed. 1 The chemical structure of quaternized chitosan was characterized by 1H NMR.

[0164] sponge preparation

[0165] Quaternized chitosan (1.5 g) and chitosan (1.5 g) were dissolved in 100 mL of 2 vol% acetic acid aqueous solution to obtain a quaternized chitosan / chitosan solution. After centrifugation, the supernatant was loaded into a mold, cooled to -20 °C, and freeze-dried to obtain QCC sponge. The QCC sponge was immersed in sodium hydroxide / ethanol solution to neutralize excess acetic acid. After thorough washing with deionized water, it was pre-cooled (-20 °C) and freeze-dried. 1 g of the dried QCC sponge was immersed in 50 mL of activation solution containing 0.4 g of reactive oxygen species-responsive hydrogen sulfide donor, 1 g of EDC, and 0.6 g of NHS. After reacting for 48 h, the sponge was thoroughly washed with deionized water, then pre-cooled to -20 °C and freeze-dried to obtain a reactive oxygen species-responsive hydrogen sulfide donor-modified QCC sponge, named reactive oxygen species-responsive controlled release of hydrogen sulfide cationic chitosan sponge, with Mw of 100 kDa.

[0166] Synthesis and characterization of reactive oxygen species-responsive hydrogen sulfide donors and quaternized chitosan

[0167] reactive oxygen species responsive hydrogen sulfide donors 1 The following 1H NMR data demonstrate the successful synthesis of a reactive oxygen species-responsive hydrogen sulfide donor: 7.74 (d, J = 8.0 Hz, 2H, -Ph-H) (a), 7.30 (d, J = 8.0 Hz, 2H, -Ph-H) (b), 3.85 (s, 2H, -Ph-CH2-S) (c), 2.65 (t, J = 6.6 Hz, 2H, -S-CH2-) (d), 2.58 (t, J = 6.6 Hz, 2H, -CH2-COOH) (e), and 1.32 (s, 12H, -C-(CH3)2) (f).

[0168] Quaternized chitosan 1 In the 1H NMR spectrum, the chemical shifts at 4.5 and 3.2 ppm are attributed to the methylene protons in chitosan and the methyl protons of the quaternary ammonium salt group in the 2,3-epoxypropyltrimethylammonium chloride molecule, respectively, proving the successful synthesis of quaternized chitosan.

[0169] Sponge preparation and performance characterization / evaluation

[0170] Figure 1 A represents the preparation process of hydrogen sulfide / QCC sponge. First, a quaternized chitosan / chitosan solution was freeze-dried to prepare the QCC sponge, where quaternized chitosan served as the bioactive component and chitosan as the cross-linking agent. ATR-FTIR results confirmed the successful composite of quaternized chitosan and chitosan. Figure 1 (Middle B). In the spectrum, 1465 cm⁻¹ -1The characteristic peaks at 1646 and 1580 cm⁻¹ belong to quaternary ammonium salt groups. -1 The characteristic peaks at these locations are attributed to amide I and amide II bonds in the chitosan molecule, respectively. Next, the QCC sponge was chemically modified using a reactive oxygen species-responsive hydrogen sulfide donor to prepare a hydrogen sulfide / QCC sponge. The modification mechanism of the sponge is an amidation reaction between the carboxyl groups in the reactive oxygen species-responsive hydrogen sulfide donor and the amino groups in the QCC sponge. 1 H NMR results confirmed the successful modification of the reactive oxygen species-responsive hydrogen sulfide donor. Figure 1 (C). In the spectrum, chemical shifts a and b at 6.5–7.0 ppm are attributed to the methylene protons of the benzene ring in the reactive oxygen species-responsive hydrogen sulfide donor, and chemical shift c at 3.23 ppm is attributed to the methyl protons of the quaternary ammonium salt group in the quaternized chitosan molecule. Furthermore, X-ray photoelectric spectroscopy (XPS) and energy dispersive spectroscopy (EDS) results further confirm the above conclusions. Compared to QCC sponges, hydrogen sulfide / QCC sponges exhibit distinct S2p and B1s peaks (…). Figure 1 (D and E), and new distributions of sulfur (S) and boron (B) ( Figure 1 In F, the scale bar is 200 μm.

[0171] The water absorption rate of hydrogen sulfide / QCC sponge is 1762 ± 119%. Figure 1 The presence of hydrogen sulfide (H2S) indicates its ability to effectively absorb wound exudate and maintain a dry healing environment. The high water absorption rate of the H2S / QCC sponge is attributed to its internal porous structure and inherent hydrophilicity. The water absorption rate of the H2S / QCC sponge is comparable to that of the QCC sponge, indicating that modification with a reactive oxygen species-responsive hydrogen sulfide donor has no significant impact on the sponge's water adsorption performance. The H2S / QCC sponge possesses a three-dimensional porous structure (…). Figure 1 The medium H (with a scale bar of 300 μm) facilitates oxygen / nutrient transport, metabolite removal, and cell migration, thereby promoting wound healing. Hydrogen sulfide / QCC sponge exhibits high compressive strength (220 kPa). Figure 1 The I-value indicates that it can withstand external stress and maintain its structural integrity during wound coverage.

[0172] In the hydrogen peroxide (H2O2) treatment group, the hydrogen sulfide / QCC sponge exhibited continuous hydrogen sulfide release, with a cumulative release of 13.9 ± 0.1 μM over 96 h. Figure 1(J). In contrast, in the phosphate-buffered saline (PBS) treatment group, the hydrogen sulfide / QCC sponge released only trace amounts of hydrogen sulfide (1.2 ± 1.2 μM). These results indicate that the hydrogen sulfide / QCC sponge can respond to reactive oxygen species (ROS) and achieve controlled, on-demand release of hydrogen sulfide. Over time, the hydrogen peroxide (H2O2) level in the hydrogen sulfide / QCC sponge group gradually decreased, while the hydrogen peroxide (H2O2) level in the QCC sponge group remained essentially constant. Figure 1 The presence of K indicates that the hydrogen sulfide / QCC sponge possesses a stronger reactive oxygen species (ROS) scavenging capacity. This strong ROS scavenging capacity of the hydrogen sulfide / QCC sponge stems from its internal structure and the consumption of hydrogen sulfide. Further evaluation was conducted on the ability of the hydrogen sulfide / QCC sponge to reduce intracellular ROS levels and increase its internal hydrogen sulfide levels. Compared to the lipopolysaccharide and QCC sponge groups, the hydrogen sulfide / QCC sponge group exhibited a significantly reduced ROS level (K). Figure 1 (L and M). Compared to the blank, lipopolysaccharide, and QCC sponge groups, the hydrogen sulfide / QCC sponge group had a significantly increased hydrogen sulfide level (L and M). Figure 1 (In L and M, the scale bar in M ​​is 100 μm). The above results indicate that hydrogen sulfide / QCC sponges can effectively reduce intracellular reactive oxygen species levels and increase internal hydrogen sulfide levels, revealing their ability to effectively eliminate oxidative stress damage in early wound healing.

[0173] Biocompatibility assessment

[0174] Biocompatibility is a key property of wound dressings, promoting wound healing by reducing tissue irritation and providing an optimal microenvironment for tissue regeneration. The color of the supernatant in the hydrogen sulfide / QCC sponge group was similar to that in the saline group. Figure 2 (A), and its hemolysis rate is less than 5% ( Figure 2 (A) indicates that the hydrogen sulfide / QCC sponge has good blood compatibility. The optical density values ​​of human umbilical vein endothelial cells (HUVECs) and Raw264.7 macrophage suspensions in the hydrogen sulfide / QCC sponge group increased with prolonged culture time, and there was no significant difference compared to other groups. Figure 2 (Figures B and C) indicate that the hydrogen sulfide / QCC sponge exhibits low toxicity to HUVECs and Raw 264.7 macrophages. Furthermore, the cell density in the hydrogen sulfide / QCC sponge group increased over time (Figures B and C). Figure 2 The D1 and D2 scale bars were both 50 μm, and almost no red fluorescent signal (dead cells) was observed, indicating that hydrogen sulfide / QCC sponges have good cell compatibility. The blood routine / blood biochemical parameters in the hydrogen sulfide / QCC sponge group remained within the normal range. Figure 2 The presence of E~H in the hydrogen sulfide / QCC sponge indicates good biocompatibility. These results demonstrate that the hydrogen sulfide / QCC sponge exhibits good biocompatibility.

[0175] Evaluation of hemostatic ability

[0176] Significant blood loss from wounds can easily lead to serious complications, highlighting the importance of hemostatic dressings. The hemostatic ability of hydrogen sulfide / QCC sponge was evaluated using a rat femoral artery injury model. Extensive blood distribution was observed on the gauze of both the untreated group and the gelatin sponge group. In contrast, less blood was observed on the gauze of the QCC sponge and hydrogen sulfide / QCC sponge groups. Figure 3 (A). After hemostasis, compared with other groups, both the QCC sponge and hydrogen sulfide / QCC sponge groups showed significantly reduced blood loss ( ). Figure 3 The presence of B indicates excellent hemostatic properties. The hemostatic mechanism of the hydrogen sulfide / QCC sponge was investigated using blood solidification index and blood cell adhesion assays. The blood solidification index of the hydrogen sulfide / QCC sponge group was significantly lower than that of the gelatin sponge group (B). Figure 3 The results of the hydrogen sulfide / QCC (C) assay revealed that it has a stronger ability to bind red blood cells. Blood cell adhesion experiments further confirmed this conclusion. Compared to gelatin sponges, more red blood cells adhered to the surface of the hydrogen sulfide / QCC sponge (C). Figure 3 The scale bar in D and D is 5 μm, and the scale bar of the inset in D is 5 μm. Furthermore, more activated platelet adhesion was observed on the hydrogen sulfide / QCC sponge surface. Figure 3 (D in the image, where the arrow indicates activated platelets). Hydrogen sulfide / QCC sponges exhibit superior in vitro procoagulant activity compared to gelatin sponges, attributed to the synergistic effect between the sponge's porous structure and quaternary ammonium groups. The porous structure effectively promotes blood adsorption and concentration; the positively charged quaternary ammonium groups can electrostatically interact with blood cells, promoting their adhesion, aggregation, and activation.

[0177] Evaluation of immune regulation capacity

[0178] Macrophages are immune cells that play a crucial role in host defense, wound healing, and immune regulation. They can polarize into pro-inflammatory M1 macrophages or anti-inflammatory, pro-healing M2 macrophages. In diabetic wounds, impaired macrophage polarization towards the M2 phenotype leads to persistent / excessive inflammation and delayed wound healing. Therefore, an effective diabetic wound dressing should possess the ability to regulate macrophage polarization towards the M2 phenotype. After co-culturing with lipopolysaccharide-stimulated Raw 264.7 macrophages, cells in the hydrogen sulfide / QCC sponge group exhibited a rounded M2 macrophage morphology, while cells in the QCC sponge group exhibited a typical M1 macrophage morphology (with multiple pseudopodia). Figure 4 In the middle A group, the scale bar is 20 μm. After co-culturing with lipopolysaccharide-stimulated Raw 264.7 macrophages, compared to the QCC sponge group, the hydrogen sulfide / QCC sponge group showed significantly decreased CD86 expression and significantly increased CD206 expression. Figure 4The scale bars for B and B are both 100 μm. Flow cytometry results further showed that after treatment with hydrogen sulfide / QCC sponges, the expression level of CD86 in lipopolysaccharide-stimulated Raw 264.7 macrophages was significantly reduced, while the expression level of CD206 was significantly increased. Figure 4 (C~E). Furthermore, compared to the lipopolysaccharide and QCC sponge groups, the hydrogen sulfide / QCC sponge group showed significantly decreased expression levels of inflammatory cytokines (IL-1β and TNF-α), while the expression level of the anti-inflammatory cytokine (CD163) was significantly increased. Figure 4 (F~H). The above results indicate that hydrogen sulfide / QCC sponges can promote macrophage polarization towards the M2 phenotype and reduce the expression of inflammatory cytokines, which is attributed to the effective scavenging of reactive oxygen species and the sustained release of hydrogen sulfide. Western blotting was used to further explore the potential mechanism by which hydrogen sulfide / QCC sponges regulate macrophage M2 phenotype polarization. Compared with the lipopolysaccharide and QCC sponge groups, the expression of M1 macrophage markers CD86 and NF-κB was significantly downregulated in the hydrogen sulfide / QCC sponge group, while the expression of M2 macrophage marker CD206 was significantly upregulated (F~H). Figure 4 (I and J). The above results collectively indicate that hydrogen sulfide / QCC sponge can promote macrophage M2 phenotypic polarization by inhibiting the activation of NF-κB in macrophages, effectively reducing the expression of inflammatory cytokines and eliminating excessive inflammation, revealing its potential as a next-generation immunomodulatory wound dressing.

[0179] Evaluation of cell migration and angiogenesis capabilities

[0180] Endothelial cell migration plays a crucial role in angiogenesis. The ability of hydrogen sulfide / QCC sponge to promote the migration of human umbilical vein endothelial cells (HUVECs) was evaluated using cell scratch and cell invasion assays. HUVECs gradually migrated towards the scratched area with increasing co-culture time. Figure 5 (A, with a scale bar of 100 μm). At 12 and 24 h, the hydrogen sulfide / QCC sponge group showed significantly improved scratch healing rates compared to other groups, at 56 ± 3% and 87 ± 4%, respectively. Figure 5 (B and C). Furthermore, the crystal violet staining intensity and cell number in the hydrogen sulfide / QCC sponge group were significantly stronger / higher than those in the other groups ( Figure 5 (D and E, with a scale bar of 100 μm in E). These results indicate that the hydrogen sulfide / QCC sponge possesses excellent ability to promote the migration of human umbilical vein endothelial cells, revealing its potential pro-angiogenic capacity.

[0181] The angiogenesis-promoting ability of hydrogen sulfide / QCC sponges was further investigated using a tube formation assay. Compared with other groups, the hydrogen sulfide / QCC sponge group exhibited more complete and numerous tubular structures. Figure 6The scale bars in groups A and B (with a scale bar of 100 μm in group A) indicate that the hydrogen sulfide / QCC sponge has a stronger pro-angiogenic capacity. Immunofluorescence staining was used to investigate the mechanism of angiogenesis in the hydrogen sulfide / QCC sponge group. Compared with other groups, the expression of vascular endothelial growth factor (VEGF) and endothelial-derived nitric oxide synthase (eNOS) was significantly upregulated in the hydrogen sulfide / QCC sponge group. Figure 6 (The scale bars in C~E are all 20μm).

[0182] Antibacterial activity evaluation

[0183] The in vitro antibacterial activity of hydrogen sulfide / QCC sponges against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) was evaluated using plate counting and live / dead fluorescence staining methods. The antibacterial rates of the hydrogen sulfide / QCC sponges against MRSA and E. coli were 89 ± 4.5% and 91 ± 2.8%, respectively. Figure 7 (A). Consistent with quantitative data, the hydrogen sulfide / QCC sponge group had fewer bacterial colony units ( Figure 7 (B). Furthermore, the hydrogen sulfide / QCC sponge group exhibits a stronger red fluorescence signal ( Figure 7 (The scale bar in C is 10 μm). The above results indicate that the hydrogen sulfide / QCC sponge has strong antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli, mainly attributed to the positively charged quaternary ammonium groups in the sponge. These quaternary ammonium groups can electrostatically interact with the negatively charged bacterial biofilm, thereby disrupting the biofilm's integrity and leading to bacterial death. The in vivo antibacterial activity of the hydrogen sulfide / QCC sponge against MRSA and Escherichia coli was further evaluated using a rat skin full-thickness defect model infected with bacteria. The number of bacterial colony units in the hydrogen sulfide / QCC sponge group was significantly less than that in the control group (…). Figure 7 The study revealed its strong in vivo antibacterial activity. It is well known that bacterial infection induces excessive infiltration of inflammatory cells and upregulation of inflammatory cytokines. A systematic evaluation of wound inflammation and the anti-infective activity of the sponge was conducted through histological analysis. Hematoxylin and eosin (H&E) staining results showed that, compared to the control group, the hydrogen sulfide / QCC sponge treatment group exhibited a smaller amount of inflammatory cell infiltration (…). Figure 7 E, and the scale bar in E is 50 μm. Immunofluorescence staining results showed that the expression levels of CD86, IL-6, and iNOS in the hydrogen sulfide / QCC sponge treatment group were significantly lower than those in the blank group. Figure 7 (The scale bars for F and F are both 50 μm). The above results indicate that hydrogen sulfide / QCC sponge can effectively reduce wound inflammation by resisting bacterial infection, revealing its potential as a next-generation anti-infective wound dressing.

[0184] Diabetic skin wound repair

[0185] The ability of hydrogen sulfide / QCC sponge to promote wound repair was evaluated using a diabetic rat model of full-thickness skin defects, and commercially available 3M... TM The dressing served as a control. Qualitative results showed that the wound area in all groups gradually decreased over time, with the hydrogen sulfide / QCC sponge group exhibiting a smaller wound area compared to the other groups. Figure 8 In Figures A and B, the scale bar in Figure A is 5mm. Quantitative results showed that the wound healing rate of all groups gradually increased over time. Among them, the hydrogen sulfide / QCC sponge group had a higher wound healing rate compared with other groups, indicating that it has a stronger ability to promote wound healing. Figure 8 (C). The excellent wound healing ability of hydrogen sulfide / QCC sponge may stem from its strong reactive oxygen species scavenging, immune regulation, antibacterial, and angiogenesis-promoting capabilities.

[0186] Seven days post-surgery, wound tissue was collected for histological analysis to investigate the wound-healing mechanism of hydrogen sulfide / QCC sponge. Hematoxylin-eosin (H&E) staining and Masson's trichrome staining results showed that, compared to 3M... TM Wounds treated with dressings and QCC sponges show more intact epidermal / dermal structure and more new collagen deposition compared to wounds treated with hydrogen sulfide / QCC sponges. Figure 8 (D and E in the middle).

[0187] Macrophages, as important immune cells, play a crucial role in early wound healing, with M2 macrophages exhibiting significant anti-inflammatory properties. Immunofluorescence staining was used to evaluate the expression levels of CD86 and CD206 to assess the level of inflammation in the wound. Compared to 3M... TM Wounds treated with dressings and QCC sponges, and wounds treated with hydrogen sulfide / QCC sponges, showed significantly reduced CD86 expression levels and significantly increased CD206 expression levels. Figure 8 The presence of F and G (with a scale bar of 50 μm in both F and G) reveals their strong anti-inflammatory capabilities.

[0188] Angiogenesis plays a crucial role in wound healing by promoting fibroblast recruitment, reducing inflammation, and delivering nutrients to the wound site. Immunofluorescence staining was used to evaluate CD31 expression levels to assess angiogenesis at the wound site. Compared to 3M... TM Wounds treated with dressings and QCC sponges, and wounds treated with hydrogen sulfide / QCC sponges, showed significantly increased CD31 expression levels. Figure 8 The H (with a scale bar of 50 μm) reveals its excellent angiogenesis-promoting ability.

[0189] In conclusion, hydrogen sulfide / QCC sponges promote the repair of diabetic skin wounds by synergistically enhancing epidermal / dermal regeneration (re-epithelialization), collagen deposition, anti-inflammation, and angiogenesis, demonstrating great potential for clinical translation and application.

[0190] In summary, the reactive oxygen species-responsive hydrogen sulfide-controlled cationic chitosan sponge provided by this invention, namely the hydrogen sulfide / QCC sponge or H2S / QCC sponge, possesses a porous structure, high mechanical strength, and strong water absorption. It can controllably release hydrogen sulfide on demand in response to reactive oxygen species, while simultaneously reducing intracellular reactive oxygen species levels and increasing internal hydrogen sulfide levels, effectively eliminating oxidative stress. By inhibiting NF-κB activation, it regulates macrophage polarization towards the M2 phenotype, thereby downregulating the expression levels of inflammatory cytokines and eliminating excessive inflammation. It enhances the migration and angiogenesis capacity of human umbilical vein endothelial cells (HUVECs) by upregulating the expression of vascular endothelial growth factor (VEGF) and endothelial-derived nitric oxide synthase (eNOS). Through synergistic enhancement of re-epithelialization, collagen deposition, anti-inflammation, and angiogenesis, it promotes the repair of diabetic skin wounds, with repair effects superior to commercially available 3M. TM Dressings. Furthermore, hydrogen sulfide / QCC sponges exhibit strong hemoconcentration and hematopoietic cell adhesion, aggregation, and activation abilities, demonstrating superior hemostatic capabilities compared to commercially available gelatin sponges in a rat femoral artery injury model. Hydrogen sulfide / QCC sponges also exhibit strong antibacterial and anti-infective activity against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli, effectively reducing wound inflammation. In addition, hydrogen sulfide / QCC sponges demonstrate good biocompatibility and biosafety. This invention provides a reactive oxygen species-responsive hydrogen sulfide-controlled-release cationic chitosan sponge (hydrogen sulfide / QCC sponge or H2S / QCC sponge) that integrates enhanced immune regulation, angiogenesis, and anti-infective functions, showing great application potential in diabetic wound repair.

[0191] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An active oxygen-responsive hydrogen sulfide donor, characterized in that, having a structure shown in Formula I: Formula I.

2. The method for preparing an active oxygen-responsive hydrogen sulfide donor according to claim 1, characterized by, comprising the following steps: reacting pinacol 4-bromomethylphenylboronate with thiourea to obtain compound 1; substituting compound 1 with 2,2'-dithiodipyridine to obtain compound 2; mercapto reaction of compound 2 with 3-mercaptopropionic acid to obtain the reactive oxygen species-responsive hydrogen sulfide donor; compound 1 has a structure shown in Formula 1, and compound 2 has a structure shown in Formula 2: Formula 1, Formula 2.

3. The use of the reactive oxygen species-responsive hydrogen sulfide donor according to claim 1 in the preparation of a wound dressing.

4. A reactive oxygen response hydrogen sulfide controlled release type cationized chitosan sponge, characterized by, having a structure shown in Formula II: Formula II, The Mw of the reactive oxygen species-responsive hydrogen sulfide controllable release type cationized chitosan sponge is 90-110 KDa.

5. The method for preparing the active oxygen-responsive hydrogen sulfide-controlled release type cationized chitosan sponge according to claim 4, characterized in that, comprising the following steps: precooling the quaternary ammonium chitosan / chitosan solution to-20 ℃, and then freeze-drying to obtain a QCC sponge; coupling a hydrogen sulfide donor to the surface of the QCC sponge through an amidation reaction to obtain the reactive oxygen species-responsive hydrogen sulfide controllable release type cationized chitosan sponge, wherein the hydrogen sulfide donor is the reactive oxygen species-responsive hydrogen sulfide donor according to claim 1.

6. The preparation method according to claim 5, characterized in that, The mass ratio of quaternary ammonium chitosan to chitosan in the quaternary ammonium chitosan / chitosan solution is 1:

1.

7. The production method according to claim 5 or 6, characterized by, The total mass of quaternary ammonium chitosan and chitosan in the quaternary ammonium chitosan / chitosan solution to the volume of the solvent is 3 g:100 mL, the solvent is an aqueous acetic acid solution, and the concentration of acetic acid in the aqueous acetic acid solution is 2 vol%.

8. The preparation method according to claim 5, characterized in that, The molar ratio of carboxyl groups in the reactive oxygen species-responsive hydrogen sulfide donor to amino groups in the QCC sponge is not less than 1.

9. The use of the reactive oxygen species-responsive hydrogen sulfide controllable release type cationized chitosan sponge according to claim 4 in the preparation of a wound dressing.

10. Use according to claim 9, characterized in that, The wound dressing is used for diabetic wound repair.

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